Study On Construction Of The Mouse EMSP1 Gene Knockout Vector And Production Of Feeder Layers Of ES Cell

Establish upon the embryo stem cell technique and gene target, genetic determinant could betransform to procure reconstructive animal model. Esteblishment of mouse model is a hot spot ofgene target, the homologous recombine ES cell clones were inject into blastocoele to gainallophenic mice, further breeding integral genetic systematic mice strain.This technique line hasapply for gene knockout mouse successfully.One forte of this technique line is that its analysis canbe progress on monoclone,and genie allophenic mice is breeded from monoclone cell. Geneknockout and culture of mouse embryonic stem cells in vitro will establish basis for the furtherstudy on the production of gene knockout mouse by nuclear transfer clone.Two pairs of primers were designed by Oligo 6.0 software and synthesized according to EMSP 1genomic sequence which had sequenced before PRSS17 sequence(10134) from GenBank. Twogene fragments of 129 strain mouse were amplified by PCR from a 5.0kb(long arm, Larm) and a1.7kb(short arm,Sarm). Larm and Sarm was cloned into pGEM^?-T vector on the multiple clonesites(MCS) and the recombination plasmid was identified by PCR, restriction endonuclease andsequence analysis. Sarm fragment with cohesive ends was then obtained by BamHâ… and Hindâ…¢double digestion; Larm fragment with cohesive ends was then obtained by Salâ… and Xbaâ… doubledigestion. The two arms were cloned into a universal gene knockout vector—pSSC-9 on eitherside of positive selective marker—Neo. The mouse enamel matrix serine gene knockoutvector(pSSC-Larm-Sarm)was identified successfully by PCR and restriction endonuclease.To establish feeder layer culture Systerm of ICR mouse embryonic fibroblast (MEF) cells forisolaton and culture of embryonic stem (ES) cells. ICR mouse fetuses of 11.5 to 14.5 days werechosen to isolate MEF by Liberase Blendzyme 3.Different concentrations of the Mitomycin Cwere used to pretreat feeder layers. The effects of feeder layers were assessed by cell morphology.MEF cells from 12.5 days pregnant ICR mouse grew better than those from others. MEFcells from2nd to 5th passage could be used to product feeder layers.The optional concentration ofmitomycin C to inhibit proliferation of 3×10⁵ pc/ml MEF was 10 mg/L for 2 hour. ICR mousecan be used for isolation of MEF to product feeder layers for ES cells.